1. Field of the Invention
The present invention broadly relates to methods for detecting binding interactions between various types of ligands and antiligands, in particular label-free detection of ligand/antiligand complex formation in a mixture without requiring separation of the components of the mixture from each other. In one implementation, the present invention relates to methods for screening ligands for those having binding affinity for an antiligand protein or biological-cell target of interest. As such, the present invention is useful within the fields of fundamental biomedical and biochemical research, especially drug discovery and medical diagnostics.
2. Description of Related Art
Proteins play a variety of key roles in biological processes and functions, including for example, functioning as catalysts, regulators of biochemical pathways, receptors, and as important elements in immune response. Given their diverse and important roles, it is not surprising that pharmaceutical researchers have viewed ligands that bind to proteins as attractive candidates for therapeutic agents. One traditional approach for drug discovery simply involved making modifications to natural regulators. As more data regarding structure function relationships became available, it became possible to engage in rational drug design using computers and x-ray structures to aid in synthesizing molecules tailored to fit the active site of an enzyme, for example. However, even using such advanced techniques, drug screening and development remained an often tedious and time-consuming process.
More recent drug discovery methods take a different approach and involve screening extremely large libraries of compounds for their ability to bind protein targets of interest. This type of approach typically begins with the identification of a potential protein target, such as a receptor. A diverse library is then prepared containing ligands to be screened for their ability to bind the target. The libraries can be random peptide libraries, carbohydrate libraries, natural product libraries, synthetic compound libraries, etc. Often the libraries are prepared using recently developed combinatorial techniques. These libraries are subsequently subjected to high throughput screening to identify ligands that bind to the target. Because the key feature of this approach is to screen a huge number of molecules, the success of this approach hinges on the ability to rapidly screen and identify ligands that do bind the target. Ligands initially identified as binding the target are then used to develop more focused libraries that are then put through the same screening process. This process of screening and preparing new focused libraries typically is repeated several times until a relatively small population of lead compounds is identified. These lead compounds are then subjected to various pharmaceutical analyses to select useful drug candidates.
Although this process is described above for proteins, it will be recognized that there are other molecules and indeed entire biological structures (such as cells or sub-cellular organelles) of biological interest. This discussion of past techniques of drug discovery is directed primarily to proteins for simplicity, as proteins have been the most common targets. It should be recognized, however, that other molecules, structures, and cells have been the target of drug discovery operations and represent alternatives relevant to the present invention.
A primary limitation of many current screening methods is that they require labeling of either the target or ligand and are unable to detect binding complexes directly unless there is some specific (and in many cases rare) interaction that can be detected directly (such as fluorescent quenching of a tyrosine at the binding site of a protein or interaction of a specific optical frequency with only one of the components present in the mixture being analyzed for binding). Further, some methods are unable to determine the strength (affinity) of a target/ligand bond, which is a prime indicator of the specificity of molecular interaction and of the ligand's potential as a drug candidate. The ability to detect the formation and strength of protein/ligand (or, more generally, antiligand/ligand) complexes in a mixture without requiring separation of the components of the mixture from each other would represent a significant advance, and would, for example, further facilitate high-throughput drug-candidate-screening techniques.
Recently, new methods and systems for detecting binding events between ligands and antiligands have been developed in the laboratories of the present inventors utilizing a system that is sensitive to the dielectric properties of molecules and binding complexes, such as nucleic acid hybridization and protein/ligand complexes (see e.g., U.S. Pat. Nos. 6,287,776 and 6,287,874 to Hefti, and PCT/US00/28491 to Chapman et al.). Signals analyzed related to dielectric properties such as the resonant frequency of the system (including the sample as part of the system) and permittivity of the sample. Permittivity is a measure a material's ability to resist the formation of an electric field within it. This material property has conventionally been used to characterize dielectrics used, for example, in the semiconductor fabrication field. However, sensitivity and precision required for measurements in the semiconductor context are of different orders of magnitude than the far higher degree of sensitivity and precision required to detect permittivity differences arising from bonding interactions on the molecular level. The noted patents and application describe how measurements can be taken and used to detect antiligand/ligand binding. In these cases signal analysis primarily comprised detecting a similarity or difference between a test signal and a known signal or comprised measuring a signal change for each of the two potential binding partners added to an otherwise identical buffer and comparing that value to the actual value detected for the combination. In the latter case, if the sum was equal to the measured value, it was understood that no interaction had taken place, while a change was understood to indicate the occurrence of an interaction (and thus a change in the signal).
However, this type of signal analysis did not allow determination of association constants of unknowns without separation of the components. For example, one embodiment described in the PCT application cited above discussed attaching an antibody to the detection region of an apparatus. Attachment in a detection region retains the antibody in that location along with any ligand that becomes bound to the antibody, while flow of solution past the detection region allows unbound ligand to move out of the detection region. The device could be exposed to several different concentrations of the analyte and the response for each concentration measured to provide a dose-response curve, using standard techniques already known in the art that involve separation of bound and unbound species.
In addition to the work that has occurred in the laboratory of the present inventors (exemplified by the publications described above), there has been at least one instance of permittivity measurements at a level of sensitivity sufficient to detect differences in permittivity between a solution of a ligand and a solution of a ligand and an antiligand (Amo et al., Biosensors & Bioelectronics, Vol. 12, No. 9–10, pp. 953–958 (1997)). However, the Amo publication does not teach how to determine binding or how to measure binding affinity since the materials tested were already known to bind. The measurements were merely an indication that a solution containing one component of a known binding pair presented a different permittivity from a solution containing both components. Amo does not attempt to determine and/or quantify ligand/antiligand binding in solution without previously knowing information about the system being investigated (e.g., prior knowledge that binding would occur).
Techniques used to detect molecular binding in solution (and especially to measure binding affinity) in the past have usually required either physical separation of bound and unbound binding pair members from each other, labeling of one or both members of the pair, or, at a minimum, selection of a measurement property that is independent of all but one of the binding pair members. For example, the early techniques that labeled one of the members (e.g., a radioactive or fluorescent label) required separation of bound and unbound materials so that they could be distinguished from each other. This can be seen symbolically by considering component A of a potential A:B binding pair that has been labeled to provide labeled A*. The following equilibrium then occurs upon mixture of the components: A*+B⇄A*:B. However, when the label * is detected in an un-separated mixture, both A* and {dot over (A)}*B are detected. The same amount of label is detected regardless of the extent of binding unless A* is separated from A*B, in which case either can be measured and used to calculate the extent of binding, since the original amount of A* added to the solution is known.
In special cases, it has been possible to select a measurement property that is independent of all but one of the binding pair members. A known example of this situation occurs when light absorption is used to follow the binding of oxygen to hemoglobin. The spectrum of hemoglobin is different from the spectrum of oxyhemoglobin, and the differences occur at wavelengths that are not measurably absorbed by oxygen or other components of the reaction mixture. Thus, separation of components is not required in order to detect binding. Absorbance increases as oxygen is added to a hemoglobin solution and then levels off as the fraction of hemoglobin converted to oxyhemoglobin approaches 1.
This latter situation can provide graphs that superficially look similar to the graphs obtained by the analysis process of the present invention (which will be described later in detail). See, for example, FIG. 15.5 of the text Principles of Physical Biochemistry by van Holde et al., Prentice Hall, New Jersey, 1998, which shows a plot of the change in some measurable parameter X graphed against total ligand concentration. However, closer analysis shows that such graphs are not obtained using data relating to bulk properties of solutions (i.e., properties related to multiple, typically all, of the components in the solution; defined later in detail; q.v.), but to a measurement related to only one component of the solution/mixture being evaluated (rather than all or at least multiple components, as occurs with measurement of bulk properties).
It would be desirable to have an effective general technique for detecting ligand/antiligand binding based on bulk-property measurements, such as permittivity measurements, of unlabeled ligand and antiligand (e.g., small molecule drug candidates and receptor proteins) in solution (1) in the absence of prior knowledge of binding and (2) that does not require detailed prior system information.